Substituted formyltetrahydropteroyl amino acids and method of preparing the same



Patented Nov. 17, 1953 2,659,726 marvel-w SUBSTITUTED FORMYLTETEAHYDRO;

' PTEROYL AMINO ACIDS AND-METHOD? or PREPARING THEASAMEa- Donna(losulich, Plainfiel Ant wan Cianamid Qomp ny,

fi m; a-cbrporation of Maiiie No-fiiavviiig. Application prila$ 1952Serial No. 281321;

dines? Mor artimariyg ic reiatst' dro-5-formyl-iQ-alkylpteroic acids-aind -methods of fii-piairiiiitfi sarxie:

A number of publications have appared witl i in the past year ortwodescribing the existence of substances that stimulate the growth in asynthetic medium of Leuconostoc citroazmitm. This unknownisubstance liasbeemdescribewas having activity similar tocr identicalswithformyltetrahydropteroylglytamic acid.l-

substance has been found to replace the folic 'acid'r'equirement It hasalso been H toxi err? of ty' ifi -1hh1b1fing growth: therefore can beuseful f nw csmuouirassdn are be prepared by reduction andforr'iriylation of certain known compounds which have been previouslyprepared and most of whicl'il have been described in the literature. Thest'aiting materialsthat may be' usjed in the process of thepreseiitiiivention to prepare 'thinew; compounds may be represented bythe following structural formula: I y

or the l glycine, alanine; sense; oi tlie' like. Since the aliphaticamino acids contain carboxylic acid groups, obviously salts of thesecompounds may also be used in the process if desired. Similarly thefinal products may be obtained in the form of 60 salts such as thealkaline earth metal salts.

I may however, be of another aminqac aspartic.

'r'iistcampcuncs of Luis presentmvemrdn; be illustrated one or theirtautomeri'cfforfiisf by, the. following stmctiifallformulaz inwhic'hbRLis' a lower allryl radical and is A an; aliphatic amino acid.radical.

The reduction of thecomp'ounds from which the new products of thepresent invention-are, prepared 'may be conductedveither witlr or withoutvthe aid of catalysts. When using; a catalytic reduction a widevariety of: solvents'may beaused. in the process including, formic acid,alcohols, glycol, acetic,- acid andothers; dependingmupon. the nature ofy the catalyticr agent. The presence? of'water in small amounts; ispermissible; The

temperature of the reduction-may occur. over widerangeoftemperatures;ofi from 0 toabout; 100 C.

A- number of: catalysts commonly =used-fin catalytic reductions: maybeemployed. in the. process, for example, platinum oxide, palladiumcn charcoal ,-\Raney nickel and others.-

In-; ge neral,; the reaction occurs rapidly and .ap.-- preciable yieldsof thetetrahydropteridine inter mediate can beobtained in as -little.time: as? 1 minutes at room temperature using-, for. example i hydrogenwith platinum oxide catalyst in-formic acid. The reduction is usuallycompletewithim a period of 30 minutesito about two hours.

After the reaction is complete the introduction of the fo'rm'yl; groupyields the desired compound which can be purified by several methods;for

example; reprecipitation' from a: solvent; chromatog raphy on absorbents [such asrnagnesiiim sili-F cate orstarch; and isolation df'the'product as the calciumor barium saltswhich arescmmem Water but havea limited solubility in aqueous alcohol? The following examples describeam'ethodfor the preparation of a 5-formyl-10- alkylpteroyl aliphaticamino acid and certainsalts thereof.

All parts are by weight unless otherwiseindicated.

Edfample 1" A solution of 24.8 parts ofil'o-methylpteroylglutamic acidin 350 parts by volume (if 98400 70 formic acid is reduced over 0.75part of platinum orside' catalyst -at 0 Ci-l The" absorptiorr -of twomoles of hydrogen is complete in an hour. The reaction mixture istreated with 35 parts by volume of acetic anhydride and allowed to standtwenty-four hours under a nitrogen atmosphere. After removal of thecatalyst by filtration the The calcium salt is prepared by dissolving12.8

parts of the above material in '70 parts .by volume of 1 N sodiumhydroxide and after adjustin'gthe' slightly turbid solution to about pH7.5, adding a solution of 3.9 parts of calcium chloride in 12.8 parts byvolume of water. To remove considerable colored impurity, the solutionis stirred for about 5 minutes with '5.1 parts of magnesium silicate.The filtered soltuion is then added slow- 1y with stirring to 420 partsby volume of ethanol. The precipitated calcium salt is filtered, washedand dried to yield 10.8 parts.

A aqueous solution of 4.3 parts of the calcium salt is placed on amagnesium silicate column (100 parts). Elution is accomplished by waterand the presence of the desired material is determined by addition of afew drops of barium chloride and excess ethanol to a portion of theeluate; a fiocculent precipitate is considered a positive test. About1800 parts by volume of eluate is collected before any flocculence isdetected in the test. A total of 1550 parts by volume is obtained whichgives a positive test. This is concentrated under reduced pressure to asmall volume and the concentrate, after being made alkaline, is filteredto remove silicates. After adjusting the pH of the filtrate to 7-8, 1.3parts by volume of 33% aqueous calcium chloride solution is added. Theresulting solution (11 parts by volume) is added, with stirring, to 65parts by volume of ethanol. After a short cooling period, the calciumsalt is filtered, washed and dried to yield 0.285 part. A total of 8.0parts of the crude calcium salt is chromatographed as indicated aboveand the products are combined to give 2.0 parts of purified calciumsalt.

- To effect further purification, 1.4 parts of the calcium salt obtainedthus is purified on a column as before, using parts of magnesiumsilicate. The eluates containing the desired material are concentratedand the calcium salt isolated as described. This calcium salt isrecrystallized twice by warming in water and diluting slowly withalcohol to give small tablets aggregated in rosettes. The dried materialweighs 0.434 part.

Example 2 3 A small portion of the calcium salt prepared in Example 1 isconverted to the free acid by on o I I l.

N E QT o H H NH \N N/ 2 H in which R is a lower alkyl radical and NHR isan aliphatic amino acid radical of the group consisting of glutamic acidand adjacent homologs and alkaline earth metal salts thereof.

2. A compound having the formula: H

' in which R is a lower alkyl radical and NHR is a glutamic acidradical.

3. 5-formyl 10 methyltetrahydropteroylglutamic acid. 7

. 4. Calcium 5 formyl 10 methyltetrahydropteroylglutamate.

5. A process of preparing a compound having reduced pteridine in the5-position and separating the said compound.

6. A process of preparing compounds having the formula:

in which R is a lower alkyl radical and NHR' is ing a 'lO-loweralkylpteroylglutamic acid to the action of hydrogen and platinum. oxideuntil two molecular equivalents of hydrogen are absorbed, formylatingthe reduced pteridine in the 5-position and separating the said compoundtherefrom. v

'7; In a process of preparing a 5-formyl-5,6,7,8- tetrahydro l0 loweralkylpteroylglutamic acid the step which comprises formylating a 5,6,73-tetrahydro 10 lower alkylpteroylglutamic acid and recovering saidcompound therefrom.

8. A process of preparing 5-formyl-10-methy1- tetrahydropteroylglutamicacid which comprises subjecting 10-methylptemylglutamic acid to theaction of hydrogen and platinum oxide until two molecular equivalents ofhydrogen are absorbed, formylating the reduced .pteridine in the5-position and separating said compound therefrom.

DONNA B. COSULICH.

1. A COMPOUND OF THE GROUP CONSISTING OF THOSE HAVING THE FORMULA:
 5. APROCESS OF PREPARING A COMPOUND HAVING THE FORMULA: